Ingrid 't Hart

98 Chapter 5 5 Results and Discussion Chemical synthesis of galactose-heptose disaccharide Heptosyl acceptor 6 , equipped with an α-anomeric aminopentyl linker, was chemically synthesized star�ng from D-mannose according to a previously reported procedure. 27,28 Galactostyl donor 5 was synthesized from D-galactose as described before (Chapter 4). 29 Heptosyl acceptor 6 was glycosylated with galactosyl donor 5 in the presence of TMSOTf to give disaccharide 7 in a yield of 66% a�er purifica�on by silica gel column chromatography (Scheme 1). Disaccharide 7 was debenzoylated, followed by cataly�c hydrogena�on over Pd(OH) 2 /C and H 2 in a mixture of MeOH/H 2 O/HOAc to remove the benzyl, Nap and Cbz protec�ng groups. Disaccharide 1 was purified by Bio-Gel P-2 size exclusion chromatography. A�er careful characteriza�on by HRMS and 1D and 2D NMR analysis disacchardie 1 was used for further enzyma�c extensions. Scheme 1. Chemical synthesis of disaccharide 7 from galactosyl donor 5 and heptosyl acceptor 6 . Deprotec�ons provided the disaccharide Galβ1,3-Hep 1 . Reagents and condi�ons: (a) TMSOTf, DCM, 4 Å MS, -10 °C to 0 °C; (b) i. NaOMe, MeOH/CH 2 Cl 2 ; ii. Pd(OH) 2 /C, H 2 , MeOH/H 2 O/HOAc. Enzyma�c synthesis of ganglioside mimics of GM3, GM2 and GM1a Enzyma�c carbohydrate synthesis is o�en performed by bacterial enzymes, since these are generally easier to express and have broader substrate specifici�es compared to similar mammalian enzymes. Various bacterial enzymes have been used to synthesize ganglio-series glycans including GM3, GM2 and GM1. 26,30 GM3 has been synthesized by the α2,3-sialyltransferases: PmST1 (M144D), PmST3 and Cst-II. 26,30,31 GM2 has been synthesized from GM1 by the β1,4-GalNAc transferase CgtA. A study dealing with examing substrate specifici�es of CgtA has indicated a requirement of a sialic acid residue, which preferen�ally should be α2,3- linked. 26 GM1 is synthesized from GM2 by the β1,3-Gal transferase CgtB. Variants of CgtB showed different preferences when tested on a series of substrates (e.g. GM2-, GalNAcα-, GalNAcβ-) linked to an 6-(fluorescein-5- carbaxamido)hexanoic acid succinimidyl ester. 32 Furthermore, we and others found that CgtB is able to transfer more than one galactosyl residue if an excess of UDP-galactose is used (Chapter 3). 30 Disaccharide 1 was extended with α2,3-linked Neu5Ac moiety to give trisaccharide 2 (Hep-GM3) using PmST1 and CMP-Neu5Ac (Scheme 2). Trisaccharide 2 was isolated in an average yield (50 %), although LC-MS analysis indicated full conversion. The average yield might have been caused by presence of salts in star�ng material 1 or OBn O O BzO OBz BzO BzO O NH CCl 3 BnO BnO NapO HO O NBnCbz OBn O O BzO OBz BzO BzO BnO BnO NapO O O NBnCbz + OH O O HO OH HO HO HO HO HO O O NH 2 a b 1, quant 7, 66% 5 6